The phosphoinositide signaling system is a widespread mechanism for transducing signals from neurotransmitters, hormones, and growth factors to intracellular processes. The focus of this application is an investigation of two recent discoveries related to phosphoinositide signaling: (1) In higher animal species (guinea pig, rabbit, rhesus monkey) and in human neuroblastoma cells, lithium increases acetylcholine-stimulated second messenger Ins(1,4,5)P3. (2) in the absence of added agonist, lithium raises extracellular glutamate in cerebrocortical slices, which, via activation of the NMDA receptor, raises Ins(1,4,5)P3. The objectives of this proposal are to learn how Li+ elevates agonist-stimulated ins(1,4,5)P3, how Li+ raises extracellular glutamate, and what are the physiological relevance and consequences for cell signaling. The effect of Li+ on synaptiC glutamate regulation will be probed by studies of uptake of glutamate by slices, glia and synaptosomes, PKC-modulated exocytosis (levels of DAG and effect of selective PKC inhibitors) and endogenous inhibitors of glutamate release. The effect of Li+ on glutamate release from synaptosomes (an isolated presynaptic model) will be determined. The connection between Li+ linked changes in glutamate and Ins(1,4,5)P3. will be explored using inhibitors of glutamate release and NMDA receptor agonists. The general hypothesis that Li+ elevates Ins(1,4,5)P3 generated by any activated receptor coupled to PLC via G proteins or via increased Ca2+ influx will be tested by measuring the effect of Li+ on Ins(1,4,5)P3 in cerebrocortical slices in the presence of a wide selection of appropriate neurotransmitters. In collaboration with Dr. Ei Terasawa at the Primate Center, glutamate release in cerebral cortex of unanesthetized and unstressed rhesus monkeys will be measured in vivo by push-pull perfusion and by microdialysis in the presence and absence of lithium. preliminary studies in this model are very promising. The release of other neurotransmitters will also be studied. The effects of lithium on Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation in subcortical structures will be further explored, primarily in the hippocampus. The effects of acute and chronic lithium on various parameters in human neuroblastoma cells, i.e., Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (chronic), number and affinities of the Ins(1,4,5)P3 receptor, DAG, and cytosolic Ca2+, will be examined. These effects will also be studied in differentiated cells, which more closely resemble adult neurons. The molecular mechanism of lithium-elevated Ins (1,4,5)P3 can best be studied in pancreas and will be further investigated; i.e., is the elevation due to increased PLC activity or inhibition of Ins(1,4,5)P3 5-phosphatase, or both? Lastly, possible down-regulation of the NMDA- and Ins(1,4,5)P3-receptors in guinea pigs chronically treated with lithium will be studied. Preliminary data suggest down-regulation of the Ins(l,4,5)P3 receptor in mice. These studies may throw light on the molecular mechanism of the therapeutic action of lithium, which in the long run may lead to medical benefit.